Multiplex proteome quantification method based on isobaric dimethyl labeling

Patent metadata

Field	Value
Publication number	US-11029316-B2
Application number	US-201515754212-A
Country	US
Kind code	B2
Filing date	Nov 20, 2015
Priority date	Aug 26, 2015
Publication date	Jun 8, 2021
Grant date	Jun 8, 2021

Abstract

A multiplex proteome quantification method based on isobaric dimethyl labeling implements dimethyl labeling of peptide N-terminal in an acidic condition and C-terminal in an alkaline condition one after another by means of Hall the property that a dimethylation reaction has different rates on an amino group at the peptide N-terminal and an amino group on a Lysine side chain at the peptide C-terminal in the acidic condition. Multiplex labeling of peptide samples is implemented by means of the organic combination of various isotope forms of a dimethyl labeling reagents. The mass-to-charge ratios in MS1 of peptides after multiplex labeling are completely the same, the mass-to-charge ratios of the fragment ions in MS2 are different, and multiplex quantitative analyses are carried out by extracting the intensity values of corresponding fragment ions in the MS2.

First claim

We claim:

1. A multiplex proteome quantification method, comprising:
digesting a protein into a plurality of peptides using a protease that cleaves at a carboxyl side of lysine; and
performing dimethyl labeling of a peptide among the plurality of peptides to produce a plurality of labeled peptides, wherein the dimethyl labeling comprises:
performing a first dimethyl labeling to a peptide N-terminal under a first acidic condition using a first dimethyl labeling reagent and the second dimethyl labeling to a peptide C-terminal on a Lysine side chain under a first alkaline condition using a second dimethyl labeling reagent to form a first labeled peptide;
performing a third dimethyl labeling to a peptide N-terminal under a second acidic condition using a third dimethyl labeling reagent and a fourth dimethyl labeling to a peptide C-terminal on a Lysine side chain under a second alkaline condition using a fourth dimethyl labeling reagent to form a second labeled peptide;
performing a fifth dimethyl labeling to a peptide N-terminal under a third acidic condition using a fifth dimethyl labeling reagent and a sixth dimethyl labeling to a peptide C-terminal on a Lysine side chain under a third alkaline condition using a sixth dimethyl labeling reagent to form a third labeled peptide,
wherein each of the first dimethyl labeling reagent, the second dimethyl labeling reagent, the third dimethyl labeling reagent, the fourth dimethyl labeling reagent, the fifth dimethyl labeling reagent, and the sixth dimethyl labeling reagent comprises a first compound selected from CH 2 O, 13 CH 2 O, CD 2 O, and 13 CD 2 O, and a second compound selected from NaBH 3 CN and NaBD 3 CN, and
the plurality of labeled peptides have a same mass-to-charge ratio;

ionizing the plurality of labeled peptides and acquiring ionized forms of the plurality of labeled peptides in MS1 of a mass spectrometer;
separating and fragmenting the ionized forms of the plurality of labeled peptides and acquiring fragment ions in MS2 of the mass spectrometer; and
performing multiplex quantitative analysis based on intensities of the fragment ions in the MS2.

2. The method according to claim 1 , wherein the plurality of labeled peptides further comprises a fourth labeled peptide, or the fourth labeled peptide and a fifth labeled peptide, or the fourth labeled peptide, the fifth labeled peptide, and a sixed labeled peptide,
wherein the first to the sixth dimethyl labeling reagents are selected from a reagent comprising 13 CH 2 O and NaBH 3 CN, a reagent comprising CD 2 O and NaBD 3 CN, a reagent comprising CD 2 O and NaBH 3 CN, a reagent comprising 13 CH 2 O and NaBD 3 CN, a reagent comprising 13 CD 2 O and NaBH 3 CN, a reagent comprising CH 2 O and NaBD 3 CN, a reagent comprising CH 2 O and NaBD 3 CN, a reagent comprising 13 CD 2 O and NaBH 3 CN, a reagent comprising 13 CH 2 O and NaBD 3 CN, a reagent comprising CD 2 O and NaBH 3 CN, a reagent comprising CD 2 O and NaBD 3 CN, and a reagent comprising 13 CH 2 O and NaBH 3 CN.

3. The analysis method according to claim 2 , comprising displaying a spectrum of each of the plurality of labeled peptides on a same spectrum of MS2;
extracting and summing up intensity values of fragment ions of a, b and y for each of the plurality of labeled peptides on the MS2;
assigning the sum of intensity values of fragment ions of a, b and y as an intensity of the corresponding labeled peptide; and comparing the intensity of each of the plurality of labeled peptides.

4. The method according to claim 2 , wherein the protein is denatured, reduced, alkylated and then is incubated by the protease.

5. The method according to claim 1 , wherein the mass spectrometer comprises an Orbitrap analyzer, or a TOF analyzer, or an FT-ICR analyzer.

6. The method according to claim 1 , wherein the protease is a Lys-C protease.

7. The method according to claim 1 , performing a first dimethyl labeling proceeds the second dimethyl labeling.

8. The method according to claim 1 , wherein, in each of the first acidic condition, the second acidic condition, and the third acidic condition, a pH value is 2.0 to 5.0 and a labeling time is 5 to 120 min.

9. The method according to claim 1 , wherein, in each of the first alkaline condition, the second alkaline condition, and the third alkaline condition, a pH value is 7.5 to 12 and a labeling time is 5 to 120 min.

Assignees

• Dalian Inst Chem & Physics Cas

Inventors

• Zhang Lihua
• Liu Jianhui
• Zhou Yuan
• Yang Kaiguang
• Zhang Yukui

Classifications

• G01N30/04

  Preparation or injection of sample to be analysed · CPC title

• G01N30/02

  Column chromatography · CPC title

• G01N30/72

  Mass spectrometers {(mass spectrometers per se H01J49/00)} · CPC title

• G01N33/6827

  Total protein determination, e.g. albumin in urine · CPC title

• G01N2458/15

  Non-radioactive isotope labels, e.g. for detection by mass spectrometry · CPC title